EP0567508A1 - Makroskopische orientierte zelladhäsionsproteine zur wundbehandlung - Google Patents
Makroskopische orientierte zelladhäsionsproteine zur wundbehandlungInfo
- Publication number
- EP0567508A1 EP0567508A1 EP92902905A EP92902905A EP0567508A1 EP 0567508 A1 EP0567508 A1 EP 0567508A1 EP 92902905 A EP92902905 A EP 92902905A EP 92902905 A EP92902905 A EP 92902905A EP 0567508 A1 EP0567508 A1 EP 0567508A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- wound
- cell adhesion
- adhesion protein
- protein
- fibronectin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
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- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- KNVAYBMMCPLDOZ-UHFFFAOYSA-N propan-2-yl 12-hydroxyoctadecanoate Chemical compound CCCCCCC(O)CCCCCCCCCCC(=O)OC(C)C KNVAYBMMCPLDOZ-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 102000003601 transglutaminase Human genes 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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- 102100036537 von Willebrand factor Human genes 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
Definitions
- the present invention relates to materials for use in promoting wound healing, to processes for their production and their use in treating wounds in humans and animals.
- Typical examples of failure include cases of severe burns involving substantial tissue damage where the wounds often do not even close completely and where skin grafts are required to secure granulation, cases of leg ulcers where, even when the wounds heal, the healed scar is physically weak and liable to break open very easily and cases where, although a wound would heal naturally, the scarring that remains may be unsightly or cause discomfort.
- Other wounds which frequently require intervention are serious bone fractures and wounds to cartilage, ligaments and tendons which heal slowly or not at all or where the healed wound will not be sufficiently strong.
- the present inventors have developed macroscopically oriented materials, comprising a cell adhesion protein such as fibronectin, which, surprisingly have been shown to promote wound healing, in particular by creating a scaffolding to which the invading cells can adhere thus facilitating this stage of the wound healing procedure. Moreover, by aligning these materials with features of the wound or surrounding tissue, cell invasion may be directed along desired orientations thereby strengthening the initial repair and reducing the amount of reorientation required during the remodelling stage. Thus wound healing may be promoted and the mature healed wound can be made stronger or more cosmetically acceptable or both.
- a cell adhesion protein such as fibronectin
- the present invention therefore provides porous macroscopically oriented cell adhesion protein.
- Cell adhesion proteins useful in the present invention include fibronectin, vitronectin and von illebrand protein (also called von Willebrand factor) .
- Fibronectin is the preferred cell adhesion protein.
- the invention further provides porous macroscopically oriented cell adhesion protein for use in methods of surgery or therapy practised on the human or animal body.
- the invention further provides the use of porous macroscopically oriented cell adhesion protein in the manufacture of medicaments, dressings or devices for use in methods of surgery or therapy practiced on the human or animal body.
- the methods of surgery or therapy involve promoting wound healing or directing wound healing or improving the appearance or strength of a healed wound or any combination of two or more thereof.
- the method of surgery or therapy may alternatively involve the growth of autograft material such as skin or ligament promoted or directed by porous macroscopically oriented cell adhesion protein.
- the present invention also provides a method of treatment of a wounded human or animal comprising applying an effective non-toxic amount of porous macroscopically oriented cell adhesion protein to the wound.
- the macroscopically oriented cell adhesion protein of the invention comprises large scale aggregates of cell adhesion protein, which self-assemble under favourable conditions as fibrils, the molecules in each individual fibril lying substantially parallel to each other, each individual fibril being oriented over a distance of at least 100 ⁇ m and the fibrils being oriented substantially parallel to each other over macroscopic distances such as at least 0.1 mm, preferably 0.5 and most preferably for at least 1 mm. Individual fibrils may show orientation over a considerable distance, for instance up to 0.5 mm, possibly up to 1 mm or even for 5 mm or more, for instance 1,2,3 or 5 cm.
- the aggregate of fibrils may be oriented for over 5 mm or 1 cm or more, for instance 2,3, or 5 cm and, when prepared as a continuous web for subsequent division into individual dressings, the aggregate may be oriented over distances of many centimetres or even many metres.
- the fibrils are oriented in a single direction and form a sheet or mat, possibly on a substrate for support, which may be applied to a wound.
- sheets or mats may be laminated in non-parallel directions, for instance with the fibrils of one layer oriented at 90° to fibrils in a second layer.
- the fibrils may be arranged into fibres or ma be formed on a substrate or oriented by fibres of a substrate, and such fibres may be formed into woven and non- woven webs having at least one and often two or more orientation directions.
- the substrate will be a biodegradable or resorbable material such that it may be left in the wound and will eventually be destroyed as the wound heals or once it has healed or the substrate may b a physical support which is removed after formation of the oriented material.
- the materials of the invention may be applied to wounds to direct and promote the cell invasion and thereby t increase the strength, cosmetic acceptability, healing time or other desirable characteristic of the healed wound.
- a simple unidirectionally oriented mat may be used with the orientation direction across the width of a linear wound in order to promote the closing of the wound an enhance the resistance to re-opening of the wound.
- more complex webs having multiple orientation directions may be used to promote regrowth of damaged tendons, intervertebral discs and corneas whilst directing adoption by the invading cells of orientations matched to that of the surrounding undamaged tissue or to recreate orientations of the original damaged tissue.
- the use of the oriented materials of the invention will often involve aligning one or more orientation directions of the material with respect to features of the wound or surrounding tissues.
- a particular application of the materials of the invention is in stimulation of new capillary growth, a frequently perceived objective for many forms of wound repair.
- one part of the process of angiogenesis is endothelial cell adhesion to and migration over the substrate matrix.
- a development of the present invention can be applied to this by promoting attachment/migration of capillary cells to discrete fibres or strands. These strands would be orientated in the direction of the required capillary growth. Strands can take the form of (i) pure fibronectin in macroscopic fibrous form; (ii) oriented Fn strands laid into conventional wound implant materials (e.g.
- gelatin or modified cellulose sponges gelatin or modified cellulose sponges
- oriented fibronectin coated on braided resorbable sutures Whether formed of oriented Fn or fibronectin coated, the individual strands should be less than 200 ⁇ wide (ideally between 1 and 100 ⁇ m.
- a chemotactic stimulus by attaching a solid, growth factor containing gel to one end of the suture.
- a solid, growth factor containing gel would be a blood or plasma clot (ideally prepared from the patient's own blood).
- Artificial substrates based on gelati (or other gel-forming material) containing the required angiogenic factor could also be used. This suture would be drawn through or across the damaged tissue in such a way that new vessels would grow towards the end bearing the gel or clot.
- suture may usefully be employed as an "angiogenic track" during repair of avascular or poorly vascular tissues such as torn menisci, ligaments or tendons.
- Fibronectin for use in accordance with the invention may be obtained commercially in non-oriented form and may be oriented by processes such as are described below.
- substantially pure fibronectin is used.
- Other cell adhesion proteins are well known in the literature; again these are available in non-oriented form and require processing for instance as described below.
- the materials of the invention will usually be provided in sterile, pyrogen-free form.
- Oriented materials according to the invention may further comprise additional therapeutic agents, for instance agents which promote wound healing such as growth factors and growth hormones, clotting factors, platelet adhesion promoters such as thrombin, agents which promote calcification, collagen, fibrinogen, antimicrobial agents and heparin.
- agents which promote wound healing such as growth factors and growth hormones, clotting factors, platelet adhesion promoters such as thrombin, agents which promote calcification, collagen, fibrinogen, antimicrobial agents and heparin.
- the fibrils and oriented materials may be used as formed or stabilised by cross-linking using chemical reagents such as glutaraldehyde or enzymes such as factor Xllla, which is a transgluta inase.
- Cross-linking with other components such as collagen and fibrinogen, for instance using a transglutaminase, is also contemplated.
- the materials of the invention include collagen and/or fibrinogen it is preferred for these also to be oriented substantially parallel to the fibrils of cell adhesion protein.
- the oriented materials of the invention are used as, or as part of, a wound dressing, or are applied to open wounds separately from a conventional dressing.
- the oriented materials are applied to the wound with the or an orientation direction aligned with features of the surrounding tissues so as to encourage invasion along the orientation direction.
- the fibres may be aligned with muscle fibres in the wound or underlying tissue, across a linear wound or parallel with or at right angles to directions in which a tissue will be strained once healed.
- the invention further provides a process for producing porous macroscopically oriented cell adhesion protein materials which process comprises forming and orienting cell adhesion protein fibrils from solution and removing the solvent.
- Solvents useful in accordance with the present process ar generally aqueous solvents such as buffered water, distilled water, demineralised water and pyrogen-free water.
- the solvent may contain additional solutes and/or suspended particles for inclusion in or deposition on the fibronectin materials.
- the solvent may be removed by evaporation, filtration concentration or by aggregating or precipitating the fibronectin using, for instance, appropriate concentrations of salts or by adjusting the pH of the solution to acidic or basic pH and collecting and drying the aggregate or precipitate.
- the oriented materials are preferably washed and dried and may be optionally stabilised, for instance by chemical cross-linking using reagents such as glutaraldehyde or enzymatically using factor Xllla.
- the cell adhesion protein may be oriented by self- association from solution, preferably a high concentration solution at 0.7mg/ml or greater, for instance greater than 1 mg/ml, such as at least 1.5 mg/ml, for instance 2 mg/ml or more or even up to 3 mg/ml or more at about neutral pH to form fibrils on solid surfaces which fibrils are sufficientl stable to be handled, recovered and dried.
- a solution at about 1.5 mg/ml is most preferred.
- a pH of about 7.6, eg using tris-HCl buffer has been found convenient.
- the solution contains soluble ionic compounds to increase the ionic strength thereof, especially in the range up to 0.5 M ionic strength.
- the solution also contains urea at preferably 1 to 3 M.
- fibronectin may be oriented by applying continuous unidirectional motion, such as by stirring, to a saturated solution and removing the solvent so as to aggregate oriented fibronectin, for instance on the stirrer. This may be recovered and blotted to form mats which may be laminated in parallel or non-parallel directions to form a lattice.
- high concentration solutions may be drawn into fibres and the solvent removed leaving fibronectin fibres comprising oriented fibrils.
- a preferred technique for drawing fibres involves dipping an applicator onto the surface of the solution and lifting the applicator to produce one or more fibres under the effects of surface tension.
- a preferred material for the applicator is the mineral mica.
- a concentrated solution of fibronectin is applied to a fibrous substrate and the solvent is removed.
- Heparin can be incorporated into the fibronectin solution (preferably at ratios of 1:5 to 1:100, by weight heparin: Fn) without impairing its ability to form strands.
- heparin ratios e.g. 1:5, heparin:Fn
- strands made with higher heparin ratios were flat, with very little mass as a result of the high level of hydration of the newly formed strands due to the heparin content.
- a solution of human plasma fibronectin purified by gelatin affinity chromatography (approx 1.0 (eg 0.5 to 1.5) mg/ml) in neutral pH buffer (10 mM phosphate or 20 mM Tris HC1 pH7.5) containing 0.15 M sodium chloride is placed into a pressurized "stirred cell” concentration device with an ultra filtration membrane (molecular weight cut off approx. 10 to 20,000 Daltons: eg Amicon PM 10 membrane).
- Such a stirred cell eg 100 ml capacity
- the volume is slowly reduced under these conditions to less than half the initial volume giving a fibronectin concentration within the cell of approx 3 mg/ml (range of 2.0 to 10 mg/ml).
- the conditions for self- aggregation will vary depending on the purity and integrity of the fibronectin starting material, but within these ranges, a large clot or mat of solid fibronectin will be formed on the stirring bar of the cell. This can be removed and fresh fibronectin solution added to permit the formation of more fibronectin matting.
- a starting solution as described in Example 1 but containing over lmg/ml of fibronectin (Fn) at a pH around neutrality and sodium chloride concentration up to 0.2M is prepared.
- a suitable flat edged "applicator” for example a 2cm glass cover slip
- This same wetted edge is now touched onto a hydrophilic surface (e.g. flat plastic culture dish) forming a small pool of the Fn solution, clinging to both the "applicator” and the surface.
- a hydrophilic surface e.g. flat plastic culture dish
- This strand of protein (spanning between surface and applicator) can be pulled across the surface for 2 to 5mm and re-attached to the surface by again touching the applicator and the surface.
- the resultant strand of protein is firmly attached to the surface by multiple subdivided fibrils at either end. They are commonly 2 to 5 ⁇ m in diameter and up to 5mm long. They are stable with or without chemical cross linking (e.g. with glutaraldehyde) and can be washed and dried without becoming dislodged. Their orientation on the "surface” can be controlled precisely.
- strands of pure fibronectin promoted a directional orientation and attachment of fibroblasts in spite of the presence of soluble fibronectin. Stands of fibronectin were still visible in such cultures after 24 hrs exposure to fibroblasts.
- Mats were prepared as described in Example 1 using a stirred cell, under a range of conditions to test for preferred composition of the starting fibronectin solution. Mat formation was assessed on the basis of the dry weight of mat recovered and uv absorbance (at 280nm) of the fibronectin solution at the start and end of the mat forming process. Fibronectin solutions made to 2M with urea were found to be preferable, giving a greater % mat formation at the same ionic strength.
- Example 3 The influence of heparin, in the starting solution of Fn, was tested on the quantity and quality of mats formed in the "stirred cell" (see Example 1). As in Example 3, the efficiency of mat production was measured as % Fn incorporated into the aggregate. Heparin was added to known concentrations of Fn solution at ratios (weight:weight) from 1:15 to 1:200 (heparin:Fn) . Heparin was from the Sigma Chemical Co., Poole, Dorset, U.K. Each mat was made under otherwise identical conditions, from solutions of Fn containing 0.1M sodium chloride, 2M urea, 50mM tris-HCl pH7.6. At ratios below 1:15 (Heparin:Fn) mat formation was largely or wholly inhibited. Beyond a ratio of 1:40 there was little change. The preferred ratio is 1:20 to 1:40. Heparin incorporation into the mat (measured by the
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9101191 | 1991-01-18 | ||
GB919101191A GB9101191D0 (en) | 1991-01-18 | 1991-01-18 | Wound treatment |
PCT/GB1992/000100 WO1992013003A1 (en) | 1991-01-18 | 1992-01-17 | Macroscopically oriented cell adhesion protein for wound treatment |
Publications (2)
Publication Number | Publication Date |
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EP0567508A1 true EP0567508A1 (de) | 1993-11-03 |
EP0567508B1 EP0567508B1 (de) | 2001-07-04 |
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ID=10688704
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Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP92902905A Expired - Lifetime EP0567508B1 (de) | 1991-01-18 | 1992-01-17 | Makroskopisch orientiertes zelladhäsionsprotein zur wundbehandlung |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP0567508B1 (de) |
JP (1) | JPH06504926A (de) |
AT (1) | ATE202787T1 (de) |
AU (1) | AU662314B2 (de) |
CH (1) | CH684271A5 (de) |
DE (1) | DE69231916T2 (de) |
DK (1) | DK0567508T3 (de) |
ES (1) | ES2085198B1 (de) |
GB (1) | GB9101191D0 (de) |
GR (1) | GR3036775T3 (de) |
WO (1) | WO1992013003A1 (de) |
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GB9315810D0 (en) * | 1993-07-30 | 1993-09-15 | Univ London | Stabilised materials |
DE4328190A1 (de) * | 1993-08-23 | 1995-03-02 | Beiersdorf Ag | Wundversorgungsartikel mit selektivem Absorptionsvermögen |
US5877149A (en) | 1995-06-07 | 1999-03-02 | Beaulieu; Andre | Deepithelialized skin diffusion cell system |
US5641483A (en) * | 1995-06-07 | 1997-06-24 | Beaulieu; Andre | Wound healing formulations containing human plasma fibronectin |
GB9724879D0 (en) * | 1997-11-26 | 1998-01-21 | Univ London | Process for the production of protein and products thereof |
GB9826658D0 (en) * | 1998-12-03 | 1999-01-27 | Univ London | Tissue repair |
JP4786095B2 (ja) * | 1999-09-15 | 2011-10-05 | ユークリッド システムズ コーポレイション | 角膜矯正レンズ装着中または装着後の角膜組織を安定化するための組成物 |
GB0031089D0 (en) * | 2000-12-20 | 2001-01-31 | Univ London | Tissue growth method and apparatus |
AU2002366585A1 (en) * | 2001-12-13 | 2003-06-23 | Andrew Stevens | Von willebrand factor-coated biocompatible implants |
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US4925924A (en) * | 1984-03-27 | 1990-05-15 | University Of Medicine And Dentistry Of New Jersey | Biocompatible synthetic and collagen compositions having a dual-type porosity for treatment of wounds and pressure ulcers and therapeutic methods thereof |
US4973466A (en) * | 1988-06-21 | 1990-11-27 | Chiron Ophthalmics, Inc. | Wound-healing dressings and methods |
GB9101183D0 (en) * | 1991-01-18 | 1991-02-27 | Univ London | Depot formulations |
JP2948708B2 (ja) * | 1991-11-27 | 1999-09-13 | ニューヨーク・ブラッド・センター・インコーポレーテッド | フィブロネクチン含有点眼液 |
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1991
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1992
- 1992-01-17 DK DK92902905T patent/DK0567508T3/da active
- 1992-01-17 EP EP92902905A patent/EP0567508B1/de not_active Expired - Lifetime
- 1992-01-17 WO PCT/GB1992/000100 patent/WO1992013003A1/en not_active IP Right Cessation
- 1992-01-17 ES ES09250038A patent/ES2085198B1/es not_active Expired - Fee Related
- 1992-01-17 AT AT92902905T patent/ATE202787T1/de not_active IP Right Cessation
- 1992-01-17 CH CH2981/92A patent/CH684271A5/de not_active IP Right Cessation
- 1992-01-17 JP JP4503559A patent/JPH06504926A/ja active Pending
- 1992-01-17 DE DE69231916T patent/DE69231916T2/de not_active Expired - Fee Related
- 1992-01-17 AU AU11722/92A patent/AU662314B2/en not_active Ceased
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See references of WO9213003A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP0567508B1 (de) | 2001-07-04 |
JPH06504926A (ja) | 1994-06-09 |
WO1992013003A1 (en) | 1992-08-06 |
AU662314B2 (en) | 1995-08-31 |
DE69231916D1 (de) | 2001-08-09 |
GB9101191D0 (en) | 1991-02-27 |
DK0567508T3 (da) | 2001-10-22 |
ES2085198A1 (es) | 1996-05-16 |
DE69231916T2 (de) | 2002-04-11 |
ATE202787T1 (de) | 2001-07-15 |
AU1172292A (en) | 1992-08-27 |
CH684271A5 (de) | 1994-08-15 |
GR3036775T3 (en) | 2002-01-31 |
ES2085198B1 (es) | 1996-12-01 |
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